Mycoplasma genitalium (MG) is a cause of urethritis in men and is becoming increasingly recognized for its etiologic role in cervicitis, endometritis, pelvic inflammatory disease, tubal factor infertility, and preterm birth in women. Unfortunately, this bacterium is resistant to cell wall-targeting antibiotics and to many of the antibiotics curretly used to treat these serious reproductive tract disease syndromes. MG infection may persist for months, and even years, in humans despite the induction of an inflammatory response and specific antibodies during infection. We and others have hypothesized that this persistence is based on the ability of MG to evade the host immune response by antigenic variation in two of its surface proteins, MgpB and MgpC located in its complex and unique terminal organelle. Supporting this hypothesis, we have shown that variation in mgpB and mgpC, the adjacent genes encoding these proteins, is extensive both in vitro and in vivo among cervical/vaginal exudates from persistently infected women and in experimentally infected primates. Despite the limited set of putative recombination genes identified in its 580 kb genome, the smallest of any self-replicating cellular organism, 4% of the MG genome is devoted to incomplete copies (termed MgPars) of mgpB and mgpC. We have shown that recombination between the sequences of mgpBC and the MgPar sites is accomplished by reciprocal segmental recombination, thus distinguishing this system from those of other bacteria in which antigenic variation is achieved by unidirectional recombination (also termed gene conversion) between the genes encoding their surface proteins and archived donor sequences. Our development of novel methods to measure the effect of environmental conditions on mgpBC/MgPar recombination, ability to construct recombination enzyme mutants in MG, and extensive experience in the study of the molecular biology and pathogenesis of this organism, predicts our successful completion of the projects proposed. We hypothesize that: (1) by sequencing a population of mgpBC variants, we will identify hot spots for recombination, preferred MgPar sequences, and flanking signature sequences associated with this process, (2) selected environmental conditions likely to be encountered in vivo will enhance the recombination rate, and (3) novel recombination enzymes, as well as those homologous to enzymes required for general recombination in other organisms, will modulate mgpBC/MgPar recombination in MG. These experiments will complement our studies (funded by other mechanisms) assessing the biologic repercussions of recombination leading to antigenic and phase variation, both in humans and in our newly developed primate model of infection. This study is significant and innovative in that the mechanisms of gene, antigenic, and phase variation will be revealed for an extremely fastidious pathogen with few recombination genes and a very limited genome. The potential impact of our focus on the molecular biology and pathogenesis of this understudied bacterium is great in that novel targets for intervention and treatment may be identified. PUBLIC HEALTH RELEVANCE: The proposed studies are focused on Mycoplasma genitalium, an emerging pathogen associated with reproductive tract (genital) disease in men and women. This organism can persist for months and even years in infected individuals, increasing the risk of sexual transmission and ascension into the upper reproductive tract to cause serious disease. Our study will examine the mechanisms used by this bacterium to vary the sequences of the proteins on its surface, allowing it to evade the host immune response to cause persistent disease in infected individuals.